Molecular cloning of genes encoding major two subunits of a eubacterial V-type ATPase from Thermus thermophilus.

The atpAB genes which encode the alpha and beta subunits of membrane ATPase from a thermophilic eubacterium, Thermus thermophilus HB8, were cloned. The deduced amino-acid sequences of the alpha subunit (583 amino acids) and the beta subunit (478 amino acids) are only moderately similar to the alpha beta subunits of the F0F1-ATPases, while they are highly similar to the major two subunits of the V-type ATPases, a family of ATPases which have been so far found in eukaryotic endomembrane vacuolar vesicles and archaebacterial plasma membranes. Thus, T. thermophilus ATPase belongs to the V-type ATPase family, even though this bacterium is a eubacterium. The hypothesis that the differentiation of an ancestral ATPase into V-type and F0F1-ATPase occurred after the evolution of a primordial cell into archaebacteria and eubacteria should be modified accordingly.

Molecular cloning and characterization of a novel protein serine/threonine kinase highly expressed in mouse embryo.

By a PCR-based screen for cDNA clones of protein kinases, we have isolated a cDNA clone encoding a novel protein kinase (referred to as EDPK) of 305 amino acids. EDPK has a catalytic domain of 271 amino acids that contains all conserved subdomains characteristic of the protein kinase family. Only short sequences are present at the N- and C-terminal ends outside the catalytic domain. EDPK expressed in Escherichia coli and in mammalian cells phosphorylated serine and threonine, but not tyrosine, residues in an exogenous substrate. The amino acid sequence similarity between EDPK and known serine/threonine kinases was less than 35%. Thus, the newly isolated protein kinase EDPK is a novel member of the serine/threonine kinase family. Northern blot analysis showed that the EDPK mRNA was highly expressed in various stages of mouse embryo development. The expression of the mRNA was also found in a variety of mouse adult tissues. These results suggest that EDPK plays a crucial role in intracellular signaling not only during mouse development but also in adult tissues.

Cloning and sequencing of a gene encoding a new member of the tetratricopeptide protein family from magnetosomes of Magnetospirillum magnetotacticum.

A gene encoding the 22 kDa protein (MAM22) which was localized in the magnetosomes isolated from the magnetotactic bacterium, Magnetospirillum magnetotacticum, was cloned and sequenced. MAM22 was composed of 220 amino acids (aa) with a molecular weight of 24,186 Da. The deduced aa sequence exhibited significant homology with a number of proteins that belong to the tetratricopeptide repeat (TPR) protein family, including mitochondrial protein import receptors and peroxisomal protein import receptors. The presence of three repeats of a degenerate 34-aa consensus sequence, suggest that MAM22 localized in magnetosome membranes may interact with the cytoplasmic proteins containing similar TPR motifs.

Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 2.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) was identified as a potent inhibitor of hepatocyte growth factor activator (HGF activator). The primary translation product of HAI-2 contains two Kunitz domains. To characterize their function, we introduced a point mutation into the reactive site of each Kunitz domain, and assayed the mutants for their HGF activator inhibitory activity. A point mutation in the COOH-terminal Kunitz domain did not affect the activity of HAI-2, whereas a point mutation in the NH2-terminal Kunitz domain markedly reduced the activity. These results suggest that the NH2-terminal Kunitz domain is mainly responsible for the HGF activator inhibitory activity of HAI-2.

A DNA fragment homologous to F1-ATPase beta subunit was amplified from genomic DNA of Methanosarcina barkeri. Indication of an archaebacterial F-type ATPase.

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.

Evaluation of hepatocyte growth factor activator inhibitor expression in normal and malignant colonic mucosa.

Gene expression of hepatocyte growth factor activator inhibitor (HAI), a recently identified Kunitz-type serine proteinase inhibitor, was analyzed in a series of human colorectal carcinoma cell lines and in human colorectal tissues. All of the 14 cell lines derived from adenocarcinoma of the colorectum expressed HAI in vitro, whereas a colon carcinoma cell line of neuroendocrine origin did not. In vivo, HAI was consistently expressed in the normal colorectal mucosa. Although the expression of HAI mRNA was conserved in adenocarcinoma tissues of the colorectum, the levels of expression were decreased in the adenocarcinoma tissues compared to the normal counterparts. There was a tendency towards an inverse correlation, albeit not well defined, between the amounts of HAI mRNA and the tumor progression. Immunohistochemical study indicated that HAI protein is present predominantly on the surface of epithelial cells of the colon and the immunoreactivity was decreased in the adenocarcinoma cells.

Diverse glycosylation of MUC1 and MUC2: potential significance in tumor immunity.

Mucins are major epithelial luminal surface proteins and function as a physical and biological barrier protecting mucous epithelia. Diverse glycosylation of mucins potentially provides a basis for tissue-specific interaction with the milieu. When mucins are associated with malignant epithelial cells, they not only protect these cells from a host environment during metastatic dissemination but also generate immunogenic epitopes which are used by the host in the detection and immunological elimination of carcinoma cells potentially depending upon their status of glycosylation. Diverse mucin structures are generated by the combination of different core peptides, of which 10 have been reported so far, multiple types of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts), and the consequences of stepwise glycosylation events. For example, the mucin 1 (MUC1) associated with malignant cells was previously believed to exhibit unique features with a lower percentage of threonine and serine residues attached to N-acetylgalactosamine and/or without extension through core 2 structures. Some of MUC1-specific monoclonal antibodies and cytotoxic lymphocytes recognize the peptide sequences PDTR within the tandem repeat portion exposed by decreased degree of glycosylation. The specific arrangement of N-acetylgalactosamine residues is shown to be generated by a combination of pp-GalNAc-Ts with different specificities. The role of core 2 branching is somewhat confusing because well-known carcinoma-associated carbohydrate epitopes such as sialyl-Le(X), sialyl-Le(a), Le(Y), and others are often expressed when O-glycans are extended through core 2 branching. The other series of well-known carcinoma-associated carbohydrate structures are truncated O-glycans, conventionally called Tn and sialyl-Tn antigens. Interestingly, these are often found to be aligned on core polypeptides, resulting in three or more consecutive truncated O-glycans. MUC2 and other mucins, but not MUC1, have unique tandem repeats containing three or more consecutive serine or threonine residues, which potentially serve as a scaffold for trimeric Tn and sialyl-Tn epitopes. We recently found, using the MUC2 tandem repeat, that trimeric Tn is a high-affinity receptor for a calcium-type lectin expressed on the surface of histiocytic macrophages. The biosynthesis of trimeric Tn was strictly regulated by the acceptor specificity of pp-GalNAc-Ts. These results strongly suggest that variation in both glycan structures and distribution of glycans on the core polypeptides give mucins unique and diverse biological functions that play essential roles in carcinoma-host and other cellular interactions.

Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor, which was identified as a potent inhibitor of hepatocyte growth factor (HGF) activator from the conditioned medium of a human carcinoma cell line. HGF activator is a blood coagulation factor XII-like serine protease that is responsible for proteolytic activation of the inactive single chain precursor of HGF in injured tissues. The predicted sequence of the primary translation product of HAI-1, which has a hydrophobic sequence in its COOH-terminal region, suggested that HAI-1 is first produced in a membrane-associated form. In this study, we identified a transmembrane form of HAI-1 integrated in the plasma membrane of cultured cells using a monoclonal antibody against HAI-1. We also identified several soluble forms of HAI-1 in the conditioned medium of the cells, indicating that multiple sites are present in the transmembrane form of HAI-1 at which proteolytic cleavage releases the extracellular domain. At least two proteases, one of which is a metalloprotease, appear to be responsible for the release. Further, the soluble forms of HAI-1 have different inhibitory activity against HGF activator. These findings suggest that proteolytic processing plays important roles in regulation of the inhibitory activity of HAI-1.

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active.

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity.

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.

Open pergolide treatment of tricyclic and heterocyclic antidepressant-resistant depression.

BACKGROUND: Recently, a dopamine hypothesis of depression was put forward, and several studies have demonstrated that direct and indirect dopamine agonists have antidepressant effects. METHODS: Using Clinical Global Impressions, we evaluated the efficacy of 4-week treatment of pergolide as an antidepressant adjuvant involving 20 unipolar depressed patients who were refractory to standard treatment with antidepressants. RESULTS: One patients (5%) were very much improved, seven (35%) much improved, four (20%) minimally improved, six (30%) no change or worse, and two (10%) not assessed. There was no significant difference in any clinical factors between the pergolide responder and non-responder group. LIMITATIONS: This study was a non-blind open trial, and pergolide was added to tricyclic and heterocyclic antidepressants. CONCLUSION: Pergolide may be useful as an antidepressant adjuvant, suggesting a potential role for dopamine-2 stimulation in the antidepressant response.

[Characteristics of medical institutions visited by patients with 26 intractable diseases who are residents of Saitama Prefecture (comparison of the data for 1984 and 1988)].

To obtain basic data concerning the availability of medical care for intractable diseases in a large city and its environs, we analyzed the data of Saitama Prefecture. These data were selected from a nationwide survey conducted by the Epidemiology of Intractable Diseases Research Committee of the Ministry of Health and Welfare in Japan. The characteristics of medical institutions visited by patients with 26 intractable diseases were analyzed. The patients were receiving financial aid for treatment. The subjects were 4234 patients in 1984 and 6804 patients in 1988. In addition, we compared the data of 1984 with those of 1988. The results are as follows: 1) In both years, the characteristics of medical institutions which were visited varied in terms of the individual disease. The proportion of patients who visited medical institutions in the same medical service area and in the same prefecture were both very low. The percentage of patients who visited medical institutions in Tokyo was more than 30% of the total, and was more than 50% for the disease with the highest proportion of patients. The dependency on Tokyo for medical institutions was inversely proportional to the distance from Tokyo. Most patients were highly dependent on a large hospital, but the proportion of patients with SMON and ulcerative colitis who visited a large hospital was markedly lower than that for other diseases. 2) A comparison of the data of 1984 with those of 1988 showed that in most of medical service areas, the proportion of patients who visited medical institutions in the same medical service area and in Saitama Prefecture increased, but in Tokyo Prefecture the proportion decreased. We continuously observed a high dependency on a university hospital located about 60km distance from downtown Tokyo, but the dependency slightly decreased in 1988. When a new branch of a university hospital opened, many intractable disease patients then depended on that branch. These results suggest that the self-sufficiency levels of medical services for intractable diseases gradually rose in Saitama Prefecture. The illness behavior of intractable disease patients in Saitama Prefecture did not show any remarkable changes, therefore those results in Saitama Prefecture might indicate a universal characteristic of medical institutions visited by intractable disease patients who live in the envirous of a large city (Tokyo).

[Relationship between factors examined at health examination and serum pepsinogen levels in healthy adults. Physical measurements, blood chemical tests, drinking and smoking].

OBJECTIVE: We measured serum pepsinogen (PG) levels in healthy adults and examined their physical measurements, blood chemical test values, current drinking and smoking to investigate relationships between these factors and levels of serum PG components (serum PG I, PG II and PG I/II ratio). SUBJECTS AND METHODS: A total of 452 male adults in their 40's, who were determined to be normal or to have only chronic gastritis by endoscopy or X-ray examination of the upper gastrointestinal tract, were studied. PG I and PG II levels in sera were measured, and their relationship with physical measurements and blood chemical test values, and also with current amounts of drinking and smoking, were examined. RESULTS: 1) Height, body weight, body surface area, GOT, GPT and creatinine were found to significantly differ according to serum PG I level; body surface area, GPT and ALP significantly differed according to serum PG II level. However, none of the factors examined showed any significant correlation with the PG I/II ratio. 2) When subjects were divided into positive and negative cases using the evaluation criteria of PG components for gastric cancer screening (determined as positive on the basis of serum PG I level < or = 70 ng/ml and PG I/II ratio < or = 3.0), proposed by Miki et al., none of the factors differed significantly between the two groups. CONCLUSIONS: 1) Serum PG levels were associated with stature, serum transaminase and creatinine. 2) Although serum PG levels were associated with several factors, the effect of physical measurements and blood chemical tests on the results of the evaluation criteria of PG components proposed by Miki et al, were not remarkable.

[Classification of mood disorders: from the viewpoint of clinical psychiatry].

Various classifications of depression have been developed up to this time. They are divided into three categories--etiological, phenomenological and multidimensional classification. The author discussed characteristics of three classifications from the viewpoint of clinical psychiatry. Etiological classifications include Kielholz's one and Pichot's one. They are useful to guide to clinical practice, but clinical features are not clearly described in them. Phenomenological classifications include ICD-10 and DSM-IV. They are practical to facilitate research and improve communication among clinicians and researchers all over the world. Although their reliability is on the increase, they leave something to be desired in regard to pursuit of etiology. The most famous multidimensional classification in Japan is Kasahara and Kimura's one. It introduces multiaxial system that includes premorbid character, precipitating factor, clinical features, response to treatment, and course. However, it is not fully grounded in empirical evidence.

[Clinical study of severe anorexia nervosa: the role of intravenous hyperalimentation therapy].

In order to understand the psychopathology of severe anorexia nervosa (AN), and determine appropriate therapeutic approaches, a clinical study was conducted on 13 patients with severe AN who were hospitalized and were treated with intravenous hyperalimentation (IVH). The patients were divided into three types based on their clinical symptoms and initiating factors: Type I (Restricting Type; "Non-dieters"), Type II (Restricting Type: "Dieters"). Type III (Binge-eating/Purging Type). The clinical features of each type were evaluated. Based on this evaluation, the basic approach and the role of IVH in the treatment of each type are described as follows. Type I: The patients experience loss of appetite and subsequently, suffer involuntary weight loss as a result of psychological or physical stresses at school and/or home. Since the patients do not intentionally restrict food intake, they cannot explain the loss of appetite. The age at onset of this type is the youngest among the three groups. The patients are introverted, passive and not good at expressing their emotions. Therefore, it is often difficult to deepen the emotional commitment further. It is possible to understand the pathology of Type I through the psychosomatic model. IVH therapy promotes benign regression for Type I patients, so that the mother-child relationship may be restored. As the therapeutic progress, the mother child relationship occasionally become ambivalent. In such a case, it is important for the treatment team to support independent activities of the patients. Type II: The patients lose weight by intentionally restricting necessary food intake for reasons such as beauty or sports. Any experience of failure in studies or sports or trouble in complex personal relations can trigger the onset of AN. Weight loss is looked as a great achievement, whereas weight gain is recognized as a serious failure of self-control. Since type II patients understand the necessity of receiving treatment, it is possible to establish a trusting relationship during therapy. Their prognosis is generally good. The psychotherapeutic approach for Type II patients is most effective in the context of a weight gain program utilizing behavior therapy. It is important for the therapist to integrate psychological approach with physiological approach using IVH, and to modify cognitive distortion and body image disturbance. Type III: The patients have regularly engaged in binge eating or purging (or both) in the progress of AN. But as they intensely fear becoming fat, they refuse to maintain a minimally normal body weight. Therefore, they exhibit recurrently inappropriate compensatory behavior in order to prevent weight gain. In the therapeutic sessions, they often become ambivalent and unstable, showing dissatisfaction and reacting strongly against their therapists. The age at onset is the oldest of the three types. The prognosis is not good in many cases. IVH therapy may be required only in life-threatening situation for Type III patients. And severe bulimic patients may require sufficient drug treatment. The patients should be trained for interpersonal relationships at the day care unit or the occupational therapy unit. And they should be encouraged to adapt to real life.

[Survey and treatment strategy of antidepressant-resistant depression].

The present study investigated the phenomenology and treatment of antidepressant-resistant depressions (ARD). To be classified a nonresponder, a patient had to have been treated adequately with two tricyclic (or tetracyclic) antidepressants (i.e., a minimum of the equivalent of 150 mg of imipramine for 4 weeks). The 34 depressed patients (25 unipolar patients and 9 bipolar patients) failed to respond to these antidepressant trials. Seventy-two % of the unipolar patients were in their first depression episodes, but 89% of the bipolar patients had 2 or more previous affective episodes. The mean duration of the current episode of depressions was 3.8 years. In 5 unipolar patients, the duration of the current depression episode was 10 years or more. The addition of lithium, bromocriptine or levothyroxine treatment to tricyclic or tetracyclic antidepressants were effective in the treatment of ARD. Levothyroxine were more effective in the treatment of the bipolar patients than the unipolar patients. Safrazine, a monoamine oxidase inhibitor, and electroconvulsive therapy were also effective in the treatment of ARD, but had the drawbacks of the high incidence of moderate or severe adverse reactions and high relapse rates, respectively. We propose a step-wise approach to the treatment of ARD, which mainly includes the pharmacotherapy and can be used in Japan.

[A clinical study on non-verbal psychotherapy in childhood and adolescence: a proposed classification and psychopathological considerations].

The author examined the therapeutic sequence of 37 child and adolescent cases of neurotic disorder in whom the non-verbal approach was successively taken for more than 6 months. The cases could be classified into two types, according to their choice of non-verbal methods: "simple type" cases, who chose only one method at all their therapeutic sessions and "mixed type" cases, who took various combinations of non-verbal methods during the period of psychotherapy. In "simple type" cases, premorbid character was usually introverted and emotionally stable. As for their family situations, their fathers were rather asthenic and their mothers often lacked emotional communication in child-rearing. Patients showed one or only few clinical symptoms. The therapeutic relationship with them was superficially calm and stable, and it was often difficult to deepen the emotional commitment further. In "mixed type" cases, on the other hand, premorbid character was emotionally often unstable. Their fathers tended to be away from home, and mothers were conspicuously overprotective and meddlesome. Their clinical symptoms were varied. In the therapeutic sessions, they often became dependent on therapists and, at the same time, ambivalent and unstable in certain conditions, showing dissatisfactions and reacting strongly against their therapists. The characteristics of these two types can be observed in relation to other clinical disorders. Regarding the development of interpersonal relationship and personality, it can be seen that the two types is derived from the different quality of the "sense of basic trust" of each patient acquired in early childhood. Based on the above findings, the author discussed a few psychopathological issues on his proposed classification.